The Increase in Circulating Levels of Pro-Inflammatory Chemokines, Cytokines, and Complement C5 in Canines with Impaired Kidney Function.

Chronic low-grade inflammation is a key contributor to the progression of kidney disease. The release of cytokines and other pro-inflammatory proteins may further contribute to detrimental kidney health by increasing interstitial edema and renal fibrosis. The aim of the present study was to investigate the inflammatory markers in canines who developed renal disease naturally and were diagnosed with renal disease either during life or following necropsy, as assessed by a veterinarian. RNA was isolated from canine blood obtained at necropsy and stored as bioarchived samples from ten canines with renal disease (9.6−14.7 yr) and ten controls (10.1−14.8 yr). At the time of death, the mean blood creatinine concentration and BUN were elevated in dogs with renal disease compared to control (both p < 0.01). Samples were assessed for changes in gene expression using the Canine cytokine RT2 Profiler PCR Array for inflammation. There was a significant increase in C-C Motif Chemokine Ligand 16 (CCL16), C-X-C Motif Chemokine Ligand 5 (CXCL5), Interleukin 16 (IL-16), and Complement Component 5 (C5) (all p < 0.05 vs. con). In addition, there was also a statistically non-significant increase in 49 genes and a down-regulation in 35 genes from a panel of total 84 genes. Pro-inflammatory genes including CCL16, CXCL5, IL-16, and C5 can all contribute to renal inflammation and fibrosis through different signaling pathways and may lead to a progressive impairment of kidney function. Blockade of their activation may be important in ameliorating the initiation and/or the progression of renal disease.

Metabolomic changes in cats with renal disease and calcium oxalate uroliths.

INTRODUCTION: There is a significant incidence of cats with renal disease (RD) and calcium oxalate (CaOx) kidney uroliths in domesticated cats. Foods which aid in the management of these diseases may be enhanced through understanding the underlying metabolomic changes. OBJECTIVE: Assess the metabolomic profile with a view to identifying metabolomic targets which could aid in the management of renal disease and CaOx uroliths. METHOD: This is a retrospective investigation of 42 cats: 19 healthy kidney controls, 11 with RD, and 12 that formed CaOx nephroliths. Cats were evaluated as adults (2 through 7 years) and at the end of life for plasma metabolomics, body composition, and markers of renal dysfunction. Kidney sections were assessed by Pizzolato stain at the end of life for detection of CaOx crystals. CaOx stone presence was also assessed by analysis of stones removed from the kidney at the end of life. RESULTS: There were 791 metabolites identified with 91 having significant (p < 0.05, q < 0.1) changes between groups. Many changes in metabolite concentrations could be explained by the loss of renal function being most acute in the cats with RD while the cats with CaOx stones were intermediate between control and RD (e.g., urea, creatinine, pseudouridine, dimethylarginines). However, the concentrations of some metabolites differentiated RD from CaOx stone forming cats. These were either increased in the RD cats (e.g., cystathionine, dodecanedioate, 3-(3-amino-3-carboxypropyl) uridine, 5-methyl-2'-deoxycytidine) or comparatively increased in the CaOx stone forming cats (phenylpyruvate, 4-hydroxyphenylpyruvate, alpha-ketobutyrate, retinal). CONCLUSIONS: The metabolomic changes show specific metabolites which respond generally to both renal diseases while the metabolomic profile still differentiates cats with RD and cats with CaOx uroliths.

Experimental Biology 2020 Meeting Abstracts.

Bone morphogenetic proteins (BMPs) are growth factors that belong to the transforming growth factor-ß (TGF-ß) superfamily, and till date 15 BMPs have been described. BMPs, first described for their role in bone and cartilage formation, also play a role in renal fibrosis in chronic kidney disease (CKD). There is evidence to indicate that in rodent models of CKD, administration of recombinant BMP1-3 increases renal fibrosis whereas administration of a BMP1-3-neutralizing antibody or BMP-7 antibody reduces renal fibrosis and preserves renal function. The aim of the present study was to investigate changes in gene expression in the renal cortex obtained from cats with kidney disease or calcium oxalate stone formers (CaOx) at necropsy, to identify BMPs associated with renal dysfunction in cats and potential fibrosis. At time of death the circulating levels of creatinine as well as symmetric dimethyl arginine (SDMA), both markers of kidney decline in cats, were significantly higher in cats with renal disease (n=11) or stone-forming cats (CaOx, n=12) when compared to controls (n=19). Using RNAseq in kidney tissue, we found a modest, but significant, increase in the expression of BMP-1 in cats with kidney disease (2.48 fold) and stone formers (1.72 fold), compared to controls (both p<0.01). While the increase in BMP-2 in CaOx cats was significant (1.46 fold; p<0.05 vs Con), the increase in cats with kidney disease was not (1.23 fold; NS). BMP2K, a BMP-2 inducible kinase, was significantly increased in both kidney disease (1.43 fold) and CaOX (1.46 fold) (both p<0.05). In contrast, a significant decrease in BMP4 was observed in both groups (<2.2 fold and 1.68 fold in kidney disease and CaOx, respectively; both p<0.001 vs Con). A decrease was also seen in CRIM 1, a protein associated with podocyte filtration function and whose reduction is associated with fibrosis, in both groups. BMP-7, whose potential therapeutic role in treating CKD and reversing fibrosis has been documented, was modestly decreased in both groups (both less than 1.5 fold) compared to controls. Given that there was an increase in all three forms of TGFß (TGFß1, TGFß2, and TGFß3), a potent initiator of renal fibrosis, in both groups, and a decline in BMP-7, an endogenous inhibitor of TGFß signaling in fibrosis, compared to controls, our results profile the BMPs potentially associated with renal fibrosis in cats that may contribute to kidney dysfunction. In summary, a nutritional therapy to slow the progression of kidney dysfunction may benefit from the inclusion of dietary ingredients that attenuate renal fibrosis in cats. SUPPORT OR FUNDING INFORMATION: This study was funded by Hill's Pet Nutrition, Inc.

Additive Effect of Resveratrol on Astrocyte Swelling Post-exposure to Ammonia, Ischemia and Trauma In Vitro.

Swelling of astrocytes represents a major component of the brain edema associated with many neurological conditions, including acute hepatic encephalopathy (AHE), traumatic brain injury (TBI) and ischemia. It has previously been reported that exposure of cultured astrocytes to ammonia (a factor strongly implicated in the pathogenesis of AHE), oxygen/glucose deprivation, or to direct mechanical trauma results in an increase in cell swelling. Since dietary polyphenols have been shown to exert a protective effect against cell injury, we examined whether resveratrol (RSV, 3,5,4'-trihydroxy-trans-stilbene, a stilbenoid phenol), has a protective effect on astrocyte swelling following its exposure to ammonia, oxygen-glucose deprivation (OGD), or trauma in vitro. Ammonia increased astrocyte swelling, and pre- or post-treatment of astrocytes with 10 and 25 µM RSV displayed an additive effect, while 5 µM did not prevent the effect of ammonia. However, pre-treatment of astrocytes with 25 µM RSV slightly, but significantly, reduced the trauma-induced astrocyte swelling at earlier time points (3 h), while post-treatment had no significant effect on the trauma-induced cell swelling at the 3 h time point. Instead, pre- or post-treatment of astrocytes with 25 µM RSV had an additive effect on trauma-induced astrocyte swelling. Further, pre- or post-treatment of astrocytes with 5 or 10 µM RSV had no significant effect on trauma-induced astrocyte swelling. When 5 or 10 µM RSV were added prior to, or during the process of OGD, as well as post-OGD, it caused a slight, but not statistically significant decline in cell swelling. However, when 25 µM RSV was added during the process of OGD, as well as after the cells were returned to normal condition (90 min period), such treatment showed an additive effect on the OGD-induced astrocyte swelling. Noteworthy, a higher concentration of RSV (25 µM) exhibited an additive effect on levels of phosphorylated forms of ERK1/2, and p38MAPK, as well as an increased activity of the Na+-K+-Cl- co-transporter-1 (NKCC1), factors known to induce astrocytes swelling, when the cells were treated with ammonia or after trauma or ischemia. Further, inhibition of ERK1/2, and p38MAPK diminished the RSV-induced exacerbation of cell swelling post-ammonia, trauma and OGD treatment. These findings strongly suggest that treatment of cultured astrocytes with RSV enhanced the ammonia, ischemia and trauma-induced cell swelling, likely through the exacerbation of intercellular signaling kinases and ion transporters. Accordingly, caution should be exercised when using RSV for the treatment of these neurological conditions, especially when brain edema is also suspected.

Ischemia-induced endothelial cell swelling and mitochondrial dysfunction are attenuated by cinnamtannin D1, green tea extract, and resveratrol in vitro.

Polyphenols possess antioxidant and anti-inflammatory properties. Oxidative stress (OS) and inflammation have been implicated in the pathogenesis of cytotoxic brain edema in cerebral ischemia. In addition, OS and pro-inflammatory cytokines also damage the endothelial cells and the neurovascular unit. Endothelial cell swelling may contribute to a leaky blood-brain barrier which may result in vasogenic edema in the continued presence of the existing cytotoxic edema. We investigated the protective effects of polyphenols on cytotoxic cell swelling in bEND3 endothelial cultures subjected to 5 hours oxygen-glucose deprivation (OGD). A polyphenol trimer from cinnamon (cinnamtannin D1), a polyphenol-rich extract from green tea, and resveratrol prevented the OGD-induced rise in mitochondrial free radicals, cell swelling, and the dissipation of the inner mitochondrial membrane potential. Monocyte chemoattractant protein (also called CCL2), a chemokine, but not tumor necrosis factor-α or interleukin-6, augmented the cell swelling. This effect of monochemoattractant protein 1-1 was attenuated by the polyphenols. Cyclosporin A, a blocker of the mitochondrial permeability transition pore, did not attenuate cell swelling but BAPTA-AM, an intracellular calcium chelator did, indicating a role of [Ca(2+)]i but not the mPT in cell swelling. These results indicate that the polyphenols reduce mitochondrial reactive oxygen species and subsequent cell swelling in endothelial cells following ischemic injury and thus may reduce brain edema and associated neural damage in ischemia. One possible mechanism by which the polyphenols may attenuate endothelial cell swelling is through the reduction in [Ca(2+)]i.

Immune-Modulating Effects of Low-Carbohydrate Ketogenic Foods in Healthy Canines.

Background: Ketogenic foods limit digestible carbohydrates but contain high fat, and have antioxidant and anti-inflammatory effects as well as improving mitochondrial function. ß-Hydroxybutyrate (BHB), 1 of the ketone bodies, reduces the proinflammatory NLR family pyrin domain containing 3 inflammasomes, as well as chemokines in cultures. Objectives: We assessed the immune-modulating effects of 2 low-carbohydrate (LoCHO) foods varying in protein and fat and compared their effects with a food replete with high-carbohydrate (HiCHO) in healthy canines. Methods: Dogs were fed control food [HiCHO; ketogenic ratio (KR: 0.46) followed by LoCHO_PROT (KR: 0.97), then LoCHO_FAT (KR: 1.63) or LoCHO_FAT followed by LoCHO_PROT. Each food was fed for 5 wk, with collections in the 5th wk; 15 wk feeding total. Gene expression for circulating inflammatory cytokines from 10 dogs was assessed using the Canine RT2 Profiler polymerase chain reaction array, and fold changes were calculated using the ΔΔCt method. Results: LoCHO_FAT significantly increased circulating ß-hydroxybutyrate compared with both HiCHO and LoCHO_PROT. When compared with HiCHO, there was a significant decrease in several proinflammatory cytokines/chemokines in LoCHO_PROT and LoCHO_FAT groups, including chemokine (C-C motif) ligand (CCL)1, CCL8, CCL13, CCL17, CCL24, chemokine (C-X3-C motif) ligand 1, chemokine (C-X-C motif) receptor 1, Interleukin-10 receptor alpha ((IL)-10RA), IL-1 receptor antagonist, IL-5, and secreted phosphoprotein 1 (all P < 0.05). Interestingly, a subset of inflammatory proteins that decreased in LoCHO_PROT but not in LoCHO_FAT included IL-33, IL-6 receptor, IL-7, IL-8, Nicotinamide phosphoribosyltransferase, and tumor necrosis factor (TNF) receptor superfamily member 11B. In contrast, the decrease in inflammatory markers in LoCHO_FAT, but not in LoCHO_PROT, included complement component 5, granulocyte colony-stimulating factor or G-CSF, interferon-γ, IL-3, IL-10RB, IL-17C, Tumor necrosis factor superfamily (TNFSF)13, TNFSF13B, and TNFSF14. Decreased concentrations of selected cytokines indicate that both low-carbohydrate foods exert an anti-inflammatory effect and provide a strong rationale for testing their efficacy in dogs with inflammatory conditions. Conclusions: Both LoCHO_PROT and LoCHO_FAT foods might be important as part of immune-modulating therapeutic nutritional strategies to reduce inflammation to maintain health in canines. Our study identifies several inflammatory genes that are reduced when fed ketogenic food that were not previously reported.

Betaine and L-Carnitine Synergistically Influence the Metabolome and Immune Response in Dogs.

This study used thirty-two dogs, which were assigned to a preferred period of 14 days and then assigned to one of the four treatment foods: control (containing no added betaine, no added L-carnitine), control with 0.5% added betaine (Treatment 2), control with no added betaine and 300 ppm added L-carnitine (Treatment 3), or control with 0.5% added betaine and 300 ppm added L-carnitine (Treatment 4). All treatment foods were fed for ninety days. Untargeted blood metabolomic analysis and immune response were measured at the beginning and end of the 90-day feeding trial. Feeding betaine increased single-carbon metabolites while decreasing many carnitine-containing metabolites. Feeding L-carnitine increased many carnitine metabolites, while the combination synergistically influenced the metabolome. The combination of betaine and L-carnitine increased the cytokines released in a Tru-culture system in response to stimulation while numerically decreasing their release when unstimulated. Therefore, the combination of dietary betaine and L-carnitine could have the dual positive effects of reducing cytokine stimulation, controlling inflammation during health, and providing a robust response to bacterial infection.

Increased dietary vitamin D was associated with increased circulating vitamin D with no observable adverse effects in adult dogs.

Introduction: There is no consensus for the optimum concentration of vitamin D, although a minimum concentration of 100 ng/mL (250 nM) of circulating vitamin D, measured as 25(OH) D, has been suggested in order to support optimal health in dogs. Few studies have examined the relationship between dietary vitamin D3 (cholecalciferol) intake and the resulting concentrations of circulating 25(OH) D in adult dogs. Recommendations for dog foods for adult maintenance report a safe upper limit of 3,200 IU vitamin D/kg on a dry matter basis. However, these recommendations were not based on studies of adult maintenance requirements. Understanding the relationship between dietary vitamin D and circulating vitamin D is necessary to utilize dietary vitamin D to influence health in dogs. Methods: Five groups of adult dogs (each n = 8) were fed food of approximately 4,000 kcal/kg containing one of the following dry matter concentrations of vitamin D for 6 months: 795.7, 3087.3, 5510.9, 7314.0, and 9992.5 IU/kg. Body weight was recorded at baseline and measured weekly, and daily food intake was recorded. Blood samples were taken at baseline and at the end of the 26-week study period. Results: There were no clinical signs of vitamin D deficiency or excess. Serum concentrations of creatinine, blood urea nitrogen, albumin, hematocrit, hemoglobin, alkaline phosphatase, phosphorus, total calcium, ionized calcium, and parathyroid hormone were maintained within reference values in all groups. Circulating 25(OH) D increased in all groups except those that consumed food with 795.7 IU/kg vitamin D, and increased in a linear and quadratic fashion in response to dietary vitamin D concentration. All of the dogs fed food with 5510.9 IU/kg vitamin D or above met or exceeded 100 ng/mL (250 nM) circulating 25(OH) D. Discussion: Dietary vitamin D was positively associated with increased circulating concentrations in concentrations up to 9992.5 IU/kg dry matter, with no observable adverse effects. Consumption of ≥5510.9 IU/kg vitamin D resulted in all dogs with at least the 100 ng/mL (250 nM) circulating concentration.

Scientific Validation of Cannabidiol for Management of Dog and Cat Diseases.

Cannabidiol (CBD) is a non-psychotropic phytocannabinoid of the plant Cannabis sativa L. CBD is increasingly being explored as an alternative to conventional therapies to treat health disorders in dogs and cats. Mechanisms of action of CBD have been investigated mostly in rodents and in vitro and include modulation of CB1, CB2, 5-HT, GPR, and opioid receptors. In companion animals, CBD appears to have good bioavailability and safety profile with few side effects at physiological doses. Some dog studies have found CBD to improve clinical signs associated with osteoarthritis, pruritus, and epilepsy. However, further studies are needed to conclude a therapeutic action of CBD for each of these conditions, as well as for decreasing anxiety and aggression in dogs and cats. Herein, we summarize the available scientific evidence associated with the mechanisms of action of CBD, including pharmacokinetics, safety, regulation, and efficacy in ameliorating various health conditions in dogs and cats.

Cats with Genetic Variants of AGXT2 Respond Differently to a Dietary Intervention Known to Reduce the Risk of Calcium Oxalate Stone Formation.

This study was completed to evaluate a genotype-specific nutritional intervention for reducing the risk of calcium oxalate stone formation. Serum metabolomic profiles and genotypes of 445 cats in the colony at Hill's Pet Nutrition, Inc (Topeka, KS, USA)were assessed in a genome-wide association study, and revealed an association between genetic variants of alanine-glyoxylate aminotransferase 2 (AGXT2) and 2-oxoarginine. The most significant single nucleotide polymorphisms (SNP) associated with 2-oxoarginine was at position chrA1:212069607, [G/A] (p < 3.687 × 10−17). This SNP explained approximately 15% of the variance in 2-oxoarginine concentrations. The distribution of genotype frequencies was 0.07 AA, 0.39 AG, and 0.54 GG, with a mean relative 2-oxoarginine concentration for each genotype of 0.45 AA, 0.92 AG, and 1.27 GG, indicating a subtractive effect of the minor allele (A). Serum concentrations of two AGXT2 substrates, symmetric/asymmetric dimethylarginines (SDMA/ADMA) and ß-aminoisobutyrate (BAIB) were also strongly associated with SNP chrA1:212069607 (p < 1.43 × 10−12 and p < 2.30 × 10−14, respectively). These two AGXT2 substrates were increased with the minor allele (A), indicating that the variant of the AGXT2 gene results in decreased aminotransferase activity. Additionally, the lifetime history of stone incidence showed that cats with the AA variant of AGXT2 SNP had a 2.515× increased incidence of stones compared with cats having the GG variant (p = 0.019). In a subsequent study assessing AGXT2 genotypes, cats (n = 10 GG, 4 AG, 9 AA) were fed control or test food (containing betaine at 0.500%, and the botanicals green tea, fenugreek and tulsi at 0.25, 0.025, and 0.0015%, respectively) in a cross-over study design. Stone risk analysis was conducted on urine samples after feeding control or test food for 28 days each. A calcium oxalate titration test (COT) was performed to assess the amount of added Ox−2 (per L) required to initiate calcium oxalate crystal formation. Cats with the GG variant of the AGXT2 SNP required more added oxalate to initiate urine crystal formation after consuming test food compared with control food, indicating a decreased risk of oxalate crystal formation in GG cats. In addition, urine oxalate concentrations showed an overall effect of test food independent of genotype (p = 0.0009), which resulted in lower oxalate concentrations after consuming test food compared with control food. These data indicate that cats with the GG-specific variant of AGXT2 should benefit from a reduced risk of calcium oxalate stone formation after consuming a betaine and botanical dietary enhancement.

Na-K-Cl cotransporter-1 in the mechanism of cell swelling in cultured astrocytes after fluid percussion injury.

Brain edema and associated increased intracranial pressure are major consequences of traumatic brain injury (TBI). An important early component of the edema associated with TBI is astrocyte swelling (cytotoxic edema). Mechanisms for such swelling, however, are poorly understood. Ion channels/transporters/exchangers play a major role in cell volume regulation, and a disturbance in one or more of these systems may result in cell swelling. To examine potential mechanisms in TBI-mediated brain edema, we employed a fluid percussion model of in vitro barotrauma and examined the role of the ion transporter Na(+)-K(+)-2Cl(-)-cotransporter 1 (NKCC1) in trauma-induced astrocyte swelling as this transporter has been strongly implicated in the mechanism of cell swelling in various neurological conditions. Cultures exposed to trauma (3, 4, 5 atm pressure) caused a significant increase in NKCC1 activity (21%, 42%, 110%, respectively) at 3 h. At 5 atm pressure, trauma significantly increased NKCC1 activity at 1 h and it remained increased for up to 3 h. Trauma also increased the phosphorylation (activation) of NKCC1 at 1 and 3 h. Inhibition of MAPKs and oxidative/nitrosative stress diminished the trauma-induced NKCC1 phosphorylation as well as its activity. Bumetanide, an inhibitor of NKCC1, significantly reduced the trauma-induced astrocyte swelling (61%). Silencing NKCC1 with siRNA led to a reduction in trauma-induced NKCC1 activity as well as in cell swelling. These findings demonstrate the critical involvement of NKCC1 in the astrocyte swelling following in vitro trauma, and suggest that blocking NKCC1 activity may represent a useful therapeutic strategy for the cytotoxic brain edema associated with the early phase of TBI.

Effect of polyphenols on oxidative stress and mitochondrial dysfunction in neuronal death and brain edema in cerebral ischemia.

Polyphenols are natural substances with variable phenolic structures and are elevated in vegetables, fruits, grains, bark, roots, tea, and wine. There are over 8000 polyphenolic structures identified in plants, but edible plants contain only several hundred polyphenolic structures. In addition to their well-known antioxidant effects, select polyphenols also have insulin-potentiating, anti-inflammatory, anti-carcinogenic, anti-viral, anti-ulcer, and anti-apoptotic properties. One important consequence of ischemia is neuronal death and oxidative stress plays a key role in neuronal viability. In addition, neuronal death may be initiated by the activation of mitochondria-associated cell death pathways. Another consequence of ischemia that is possibly mediated by oxidative stress and mitochondrial dysfunction is glial swelling, a component of cytotoxic brain edema. The purpose of this article is to review the current literature on the contribution of oxidative stress and mitochondrial dysfunction to neuronal death, cell swelling, and brain edema in ischemia. A review of currently known mechanisms underlying neuronal death and edema/cell swelling will be undertaken and the potential of dietary polyphenols to reduce such neural damage will be critically reviewed.

Botanicals Reduce Circulating Concentrations of Cholesterol and Triglycerides and Work Synergistically With Arachidonic Acid to Reduce Inflammatory Cytokines in Cats.

Forty Eight cats were used to measure the effects of feeding a traditional adult cat food supplemented with either arachidonic acid (ARA), a botanical mix (botanicals) or both on circulating biochemical parameters and inflammatory cytokines. The cats were healthy adults (mean age, 3.0; range, 1.3-6.4 years). The adult cats were fed one of four foods (n = 12 per group) for 84 days (dietary changes reported as fed): a traditional adult cat food (control, 0.05% ARA no added botanicals), or control food supplemented with arachidonic acid from chicken liver (0.13% ARA when supplemented), control food supplemented with botanicals (green tea 0.5%, fenugreek 0.05%, and tulsi 0.003%), and control plus ARA (0.13% as fed) with botanicals (green tea 0.5%, fenugreek 0.05%, and tulsi 0.003%). Response variables were compared between treatments: initially, and at 84 days (end of study). The measurements were standard complete blood counts and chemistries as well as circulating cytokines. Botanical inclusion reduced (P < 0.05) circulating cholesterol and triglycerides while arachidonic acid increased (P < 0.05) their concentrations. The pro-inflammatory cytokines MCP-1, TNFα, SDF-1, Flt3L, IL-8, IL-12p40, IL-13, and IL-18 were all reduced (P < 0.05) in cats after consuming the ARA + botanicals food for 84 days with little change after consuming the other foods. Therefore, this combination of ARA and botanicals may be of value in reducing inflammation.

Dietary Carnitine and Carnosine Increase Body Lean in Healthy Cats in a Preliminary Study.

The need to maintain body lean as cats age is shown in both health and disease. In healthy cats, body lean is associated with enhanced movement and overall longevity. In many disease states (i.e., renal disease, obesity), an enhanced or minimally maximal support of body lean is associated with quality of life and is a nutritional goal in aiding in the management of the disease. This study was designed to investigate the effect of these two dietary components and their combination on body composition and circulating factors of health, including metabolomics analysis and cytokine concentration. The foods that were fed for 169 days to four groups of cats and consisted of control food (formulated to meet the nutritional needs of all adult cats), carnitine-enhanced food (control food plus 300 mg/kg L-carnitine), carnosine-enhanced food (control food plus 1000 mg/kg carnosine), and food enhanced with both (control plus 300 mg/kg carnitine and 1000 mg/kg carnosine). Dietary enhancement with L-carnitine and carnosine increased body lean at the end of the study compared to the cats consuming the control food or the combination food. The cats consuming L-carnitine alone had a decreased concentration of circulating cytokines, while those consuming the combination food had an increased concentration of glucose, pyruvate, succinate, and circulating cytokines.

Asiatic acid, a pentacyclic triterpene from Centella asiatica, is neuroprotective in a mouse model of focal cerebral ischemia.

Asiatic acid, a triterpenoid derivative from Centella asiatica, has shown biological effects such as antioxidant, antiinflammatory, and protection against glutamate- or beta-amyloid-induced neurotoxicity. We investigated the neuroprotective effect of asiatic acid in a mouse model of permanent cerebral ischemia. Various doses of asiatic acid (30, 75, or 165 mg/kg) were administered orally at 1 hr pre- and 3, 10, and 20 hr postischemia, and infarct volume and behavioral deficits were evaluated at day 1 or 7 postischemia. IgG (blood-brain barrier integrity) and cytochrome c (apoptosis) immunostaining was carried out at 24 hr postischemia. The effect of asiatic acid on stress-induced cytochrome c release was examined in isolated mitochondrial fractions. Furthermore, its effects on cell viability and mitochondrial membrane potential were studied in HT-22 cells exposed to oxygen-glucose deprivation. Asiatic acid significantly reduced the infarct volume by 60% at day 1 and by 26% at day 7 postischemia and improved neurological outcome at 24 hr postischemia. Our studies also showed that the neuroprotective properties of asiatic acid might be mediated in part through decreased blood-brain barrier permeability and reduction in mitochondrial injury. The present study suggests that asiatic acid may be useful in the treatment of cerebral ischemia.

Green tea polyphenols attenuate glial swelling and mitochondrial dysfunction following oxygen-glucose deprivation in cultures.

Astrocyte swelling is a major component of cytotoxic brain edema in ischemia. Oxidative stress and mitochondrial dysfunction have been hypothesized to contribute to such swelling in cultures. We investigated the protective effects of polyphenol-rich green tea extract (GTE) on key features of ischemic injury namely cell swelling, nitric oxide (NO) production, and depolarization of the inner mitochondrial membrane potential (Deltapsi(m)). C6 glial cultures were subjected to 5-h oxygen-glucose deprivation (OGD) and cell volume was determined using the 3-O-methyl-glucose method. At 90 min after the end of OGD, cell volume increased by > 33% and this increase was attenuated by GTE but not by the individual polyphenol components including catechin, epicatechin, or epigallocatechin gallate (EGCG). However, a combination of catechin, epicatechin and EGCG prevented swelling. OGD-induced increase in NO was further increased by GTE. OGD-induced decline in Deltapsi(m) was also attenuated by green tea extract, EGCG and a combination of catechin, epicatechin and EGCG but not by catechin or epicatechin alone. Our findings indicate a protective effect of GTE in cell swelling in ischemic injury and such protective effects may be mediated by its effect on the mitochondria. It appears that effects on cell swelling are mediated by the concerted action of more than one of the individual components of GTE.

Trauma-induced cell swelling in cultured astrocytes.

Brain edema and associated increased intracranial pressure are major consequences of traumatic brain injury that account for most early deaths after traumatic brain injury. An important component of brain edema after traumatic brain injury is astrocyte swelling (cytotoxic edema). To examine the pathophysiologic mechanisms of trauma-induced astrocyte swelling, we used an in vitro fluid percussion trauma model. Exposure of cultured rat astrocytes to 5 atm of pressure resulted in significant cell swelling at 1 to 24 hours posttrauma that was maximal at 3 hours. Because oxidative/nitrosative stress, mitochondrial permeability transition (mPT), and mitogen-activated protein kinases (MAPKs) have been implicated in astrocyte swelling in other neurologic conditions, we examined their potential roles in this model. We previously showed increased free radical generation after in vitro trauma and show here that trauma to astrocytes increased the production of nitric oxide. Trauma also induced mPT and increased phosphorylation (activation) of MAPKs (extracellular signal-regulated kinase 1/2, c-Jun-N-terminal kinase, and p38-MAPK); these changes were diminished by antioxidants and the nitric oxide synthase inhibitor N-nitro-l-arginine methyl ester. Antioxidants, N-nitro-l-arginine methyl ester, the mPT inhibitor cyclosporin A, and inhibitors of MAPKs all significantly diminished trauma-induced astrocyte swelling. These findings demonstrate that direct mechanical injury to cultured astrocytes brings about cell swelling, and that blockade of oxidative/nitrosative stress, mPT, and MAPKs significantly reduce such swelling.

NFkappaB in the mechanism of ammonia-induced astrocyte swelling in culture.

Astrocyte swelling and brain edema are major neuropathological findings in the acute form of hepatic encephalopathy (fulminant hepatic failure), and substantial evidence supports the view that elevated brain ammonia level is an important etiological factor in this condition. Although the mechanism by which ammonia brings about astrocyte swelling remains to be determined, oxidative/nitrosative stress and mitogen-activated protein kinases (MAPKs) have been considered as important elements in this process. One factor known to be activated by both oxidative stress and MAPKs is nuclear factor kappaB (NFkappaB), a transcription factor that activates many genes, including inducible nitric oxide synthase (iNOS). As the product of iNOS, nitric oxide (NO), is known to cause astrocyte swelling, we examined the potential involvement of NFkappaB in ammonia-induced astrocyte swelling. Western blot analysis of cultured astrocytes showed a significant increase in NFkappaB nuclear translocation (a measure of NFkappaB activation) from 12 h to 2 days after treatment with NH(4)Cl (5 mM). Cultures treated with anti-oxidants, including superoxide dismutase, catalase, and vitamin E as well as the MAPKs inhibitors, SB239063 (an inhibitor of p38-MAPK) and SP600125 (an inhibitor of c-Jun N-terminal kinase), significantly diminished NFkappaB activation by ammonia, supporting a role of oxidative stress and MAPKs in NFkappaB activation. The activation of NFkappaB was associated with increased iNOS protein expression and NO generation, and these changes were blocked by BAY 11-7082, an inhibitor of NFkappaB. Additionally, ammonia-induced astrocyte swelling was inhibited by the NFkappaB inhibitors, BAY 11-7082 and SN-50, thereby implicating NFkappaB in the mechanism of astrocyte swelling. Our studies indicate that cultured astrocytes exposed to ammonia display NFkappaB activation, which is likely to be a consequence of oxidative stress and activation of MAPKs. NFkappaB activation appears to contribute to the mechanism of ammonia-induced astrocyte swelling, apparently through its up-regulation of iNOS protein expression and the subsequent generation of NO.

Overexpression of Cdk5 or non-phosphorylatable retinoblastoma protein protects septal neurons from oxygen-glucose deprivation.

Activation of cyclin dependent kinases (Cdks) contributes to neuronal death following ischemia. We used oxygen-glucose deprivation (OGD) in septal neuronal cultures to test for possible roles of cell cycle proteins in neuronal survival. Increased cdc2-immunoreactive neurons were observed at 24 h after the end of 5 h OGD. Green fluorescent protein (GFP) or GFP along with a wild type or dominant negative form of the retinoblastoma protein (Rb), or cyclin-dependent kinase5 (Cdk5), were overexpressed using plasmid constructs. Following OGD, when compared to controls, neurons expressing both GFP and dominant negative Rb, RbDeltaK11, showed significantly less damage using microscopy imaging. Overexpression of Rb-wt did not affect survival. Surprisingly, overexpression of Cdk5-wild type significantly protected neurons from process disintegration but Cdk5T33, a dominant negative Cdk5, gave little or no protection. Thus phosphorylation of the cell cycle regulator, Rb, contributes to death in OGD in septal neurons but Cdk5 can have a protective role.

The Benefit of Anti-Inflammatory and Renal-Protective Dietary Ingredients on the Biological Processes of Aging in the Kidney.

One of the significant organ systems which decline in aging is the kidney. While the causes of age-associated decline in renal function are likely multifactorial, oxidative stress and inflammation are hypothesized to play important roles in the structural and functional changes of the kidney. During aging there is a general decline in the glomerular filtration rate (GFR), a primary measurement used to assess kidney function. Inflammation and oxidative stress have been hypothesized to have a significant detrimental effect on renal function in aging and this may be attenuated by renal protective dietary ingredients. These dietary ingredients may affect renal function directly or through a microbiome-mediated secondary product. Likewise, structural changes including renal tubular atrophy, interstitial fibrosis, and glomerulosclerosis have all been described in aging. Such detrimental changes may benefit from dietary ingredients that may delay or attenuate the occurrence of such changes. This review will describe the physiology and pathophysiology of aging in renal function with an emphasis on dogs and cats that develop a decline in kidney function naturally. In addition, the varying biomarkers of health and renal dysfunction will be discussed. Finally, we will evaluate the aid in the management of this normal decline through dietary intervention in animal models.